Expansion of human trophoblastic spheroids is promoted by decidualized endometrial stromal cells and enhanced by heparin-binding epidermal growth factor-like growth factor and interleukin-1 ß.

Successful pregnancy in humans depends on deep invasion of the maternal decidua by extravillous trophoblast cells (EVTs), a process regulated by autocrine and paracrine signals in the decidual-trophoblast microenvironment. Here we examined whether trophoblast invasion is affected by decidual differentiation of endometrial stromal cells (ESC) and modulated locally by cytokines and growth factors. Trophoblast spheroids were generated from the EVT-derived cell line AC-1M88 and placed onto monolayers of either undifferentiated or decidualized ESC, or directly onto tissue culture surface. Co-cultures were treated with epidermal growth factor (EGF), hepatocyte growth factor, heparin-binding EGF-like growth factor (HB-EGF), interleukin-1ß (IL-1ß) and leukaemia inhibitory factor (LIF). Expansion of spheroids over 2-3 days was significantly enhanced by a monolayer of undifferentiated ESC compared with tissue culture surface and further increased if ESC had been decidualized. HB-EGF and IL-1ß, alone or in combination with LIF, stimulated spheroid expansion but only on undifferentiated ESC. CEACAM1, an adhesion molecule implicated in trophoblast invasion, was up-regulated in AC-1M88 cells by conditioned medium from decidualized ESC, and by HB-EGF, IL-1ß and LIF in combination. Treatment of ESC with HB-EGF or IL-1ß increased the level of the tetraspanin CD82, a metastasis suppressor found in decidual cells at the implantation site. We suggest that decidualized ESC support trophoblast invasion by paracrine signals that may include HB-EGF, IL-1ß and LIF.

Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b.

BACKGROUND: Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC) decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. METHODS: Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. RESULTS: St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82). Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. CONCLUSION: St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural progesterone. St-T1b cells therefore serve as a useful model for primary ESC.

Systematic expression analysis and antibody screening do not support the existence of naturally occurring progesterone receptor (PR)-C, PR-M, or other truncated PR isoforms.

Functional progesterone withdrawal associated with human parturition has been ascribed to various mechanisms modulating the function of the classical progesterone receptors (PRs), B and A, in utero. These include up-regulation of the inhibitory PR-C isoform, described as a 60-kDa protein occurring from translation initiation at codon 595. Our initial attempts to detect PR-C yielded uninterpretable results. To systematically validate antibodies for immunodetection of PR isoforms, we generated expression vectors for PR variants originating from putative start codons AUG-289, -301, -595, -632, and -692 in addition to those for PR-B and PR-A, and for alternative splice variants PR-T, PR-S, and PR-M. All constructs were subjected to in vitro and in vivo translation and immunoblotting with a panel of 13 PR antibodies. Antibodies raised against full-length PR were generally not capable of detecting N-terminally truncated forms, whereas C-terminal antibodies did not or only weakly reacted with PR-B and PR-A but produced prominent nonspecific signals. Thus, immunodetection of N-terminally truncated PR isoforms is prone to artifacts. Proteins of about 64 kDa were expressed from PR-289 and -301, but no corresponding endogenous forms were observed. PR-T, PR-S, and PR-M cDNAs yielded no detectable translation products. No protein was translated from AUG-595 in our PR-C expression vector unless a Kozak sequence was introduced, and the product was not 60 but 38 kDa in size. Thus, the 60-kDa protein called PR-C does not originate from AUG-595 and is not a naturally occurring PR isoform.

HE6/GPR64 adhesion receptor co-localizes with apical and subapical F-actin scaffold in male excurrent duct epithelia.

A role for HE6/GPR64 in male excurrent ducts in the regulation of water balance was suggested from targeted gene mutation in the mouse. Results of the present immunolocalization study strengthen this hypothesis. Employing monospecific antibodies and laser confocal microscopy, we studied the localization of the receptor protein in the human and wild-type mouse ductuli efferentes and epididymis. We show that HE6/GPR64 is specifically associated with cell types and subcellular domains involved in the process of fluid reabsorption. In the mouse, dual labelling with anti-tubulin antibodies revealed that HE6/GPR64 was absent from the (kino-) cilia of ciliated cells. Instead, the receptor protein accumulated in the non-ciliated principal cells. Specifically, strong immunofluorescence was observed in the apical compartment of these cells. Dual labelling with phalloidin and anti-ezrin antibodies revealed that in the mouse the bulk amount of HE6/GPR64 protein co-localized with the F-actin-ezrin scaffold in brush border-like microvilli of ductuli efferentes and long stereocilia of the epididymis proper. In the ductuli efferentes, HE6/GPR64 also co-localized with the subapical F-actin network immediately below the microvilli. Comparable immunostaining patterns were observed in human and mouse; however, a specific feature of the human ductuli efferentes was an intense HE6/GPR64-related labelling of crypt-like grooves or furrows of hitherto unknown function.

Functional characterization of male germ cell-specific CREM isoforms.

Due to alternative promoter usage, splicing, and translational initiation, expression of the cAMP-responsive element modulator (CREM) gene results in the production of functionally different CREM proteins with either activating or repressing potential on target gene expression. Recently, we demonstrated 2 novel isoforms (CREM-2-F-G-H-Ib and CREM-2-G-H-Ib) in various germ cell types during normal and impaired human spermatogenesis. In contrast to known isoforms, these exhibit a transactivation domain but lack a kinase-inducible domain (KID) domain resulting in a disruption of the open reading frame. In the present study, we functionally analyzed these isoforms. Investigation of both in vitro and in vivo expressed proteins from human testis RNA suggests that a novel upstream open reading frame in exon 2 is translated from isoform CREM-2-F-G-H-Ib, giving rise to a full-length protein. Furthermore, in both isoforms, usage of downstream adeninethymine-guanines (ATGs) for translation initiation could be observed. Sequence-specific DNA binding of CREM isoforms was confirmed by electrophoretic mobility shift assays. Luciferase reporter gene assays in cells transfected with novel CREM cDNAs demonstrated that protein kinase A dependent stimulation was inhibited by coexpression of CREM-2-F-G-H-Ib but not of CREM-2-G-H-Ib.

Immortalization by large T-antigen of the adult epididymal duct epithelium.

The SV40 large T-antigen has been widely used to convert various cell types to a transformed phenotype, and also to induce progressive tumours in transgenic animals. The objectives of this review are to compare and discuss three different approaches to generate epididymal epithelial cell lines using the large T-antigen. In the first approach, retroviral transfection of primary cultures was used to immortalize canine epididymal cells in vitro; the other two approaches used transgenic mice expressing the large T-antigen. In one of these in vivo approaches, a construct consisting of the coding sequence of a temperature sensitive (ts) SV40 large T-antigen was inserted in a mouse genome. When the cells are exposed to the permissive temperature of 33 degrees C, functional expression of the large T-antigen occurs and cells start to proliferate. In the second in vivo approach a tissue-specific promoter, the 5kb GPX5 promoter, was used to direct expression of the large T-antigen to the epididymal duct epithelium.

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors.

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site.

Expression of the metastasis suppressor KAI1 in decidual cells at the human maternal-fetal interface: Regulation and functional implications.

At the human maternal-fetal interface, the decidua forms a dense matrix that is believed to limit trophoblast invasion. We investigated whether the metastasis suppressor KAI1 (CD82) is expressed at the maternal-fetal interface. Immunohistochemistry showed strong expression of KAI1 in decidual cells, whereas trophoblast cells were negative for KAI1. In luteal phase endometrium, KAI1 was present in decidualizing endometrial stromal cells. We investigated whether KAI1 expression in endometrial stromal cells is regulated by the decidualizing stimuli cAMP and progesterone or by the cytokine interleukin (IL)-1beta. Western blot analysis revealed induction of KAI1 protein by cAMP analog, but not by progesterone, in a delayed fashion. In contrast, IL-1beta rapidly stimulated KAI1 expression at the transcript level and at the protein level. Cultured decidual cells from term placenta expressed a basal level of KAI1 protein that was elevated on cAMP stimulation. Silencing of KAI1 by RNA interference attenuated expression of decorin, a decidual product implicated in limiting trophoblast invasion. This study shows for the first time the expression of KAI1 in decidual cells at the human maternal-fetal interface, where the metastasis suppressor might participate in intercellular communication with trophoblast cells and the control of trophoblast invasion.

HE6, a two-subunit heptahelical receptor associated with apical membranes of efferent and epididymal duct epithelia.

Human Epididymis-specific protein 6 [HE6 (GPR64)] is a highly conserved, tissue-specific seven-transmembrane receptor of the human epididymis. The rodent counterparts were cloned and 5'-inverse PCR employed to confirm that the cDNA sequences were full length. Downstream from the highly conserved signal peptide-coding sequence, the 5'-regions contained at least six mini-exons of less than 50 nucleotides. Multiple splice variants involving these mini-exons were cloned in the human, the majority of which was also found in rodents. Northern blot analysis showed that the tissue distribution of the mRNA was very similar in human and rodents. The human HE6 gene was assigned to the X chromosome in a region, which is syntenic to the mouse. The HE6 sequence predicted a two-subunit receptor of the LNB-TM7 subfamily. A membrane preparation and protein solubilization method was adopted to identify the endogenous epididymal proteins. Two sets of peptides were chosen for antibody production, assuming that protein scission had occurred within the conserved GPS-motif. Western blot analysis revealed abundant two-subunit proteins in human and rodents, comprising an approximately 180 kDa hydrophilic ectosubunit and a <40 kDa hydrophobic endosubunit. Deglycosylation experiments showed that the large ectosubunits were highly glycosylated, the carbohydrate side chains dramatically increasing the apparent molecular mass. Immunohistochemical studies revealed that both subunits were associated with apical membranes of efferent ductule and proximal epididymal duct epithelia.